The major objectives of the proposed research are to illuminate the mechanisms controlling the transition from specification to differentiation and to investigate the switches that drive cells from pluripotent to committed differentiated states, using the development of the C. elegans endoderm as a model system. A regulatory cascade that specifies endoderm development in the early embryo includes two redundant mesendoderm- specifying GATA-type transcription factors, their targets, the endoderm-specifying END-1/3 GATA factors, and three downstream GATA factors, all of which show functional requirements in gut differentiation and function. Initial studies revealed substantial genetic interactions between these factors and suggest that some may share functions common to both specification and differentiation of the endoderm. When broadly expressed, END-1 and -3 can reprogram non-endodermal cells into an endoderm differentiation pathway; however, the ability of cells to be thus reprogrammed is temporally confined to early stages of embryogenesis, beyond which cells appear to switch from pluripotency to committed differentiation. Functional genomics screens for genes required for this switch in early embryos and in the adult germline implicate cell fate regulating transcription factors, chromatin modification, and cell-cell communication in directing commitment of differentiating cells. The mechanisms that make cells in the embryo and developing germline refractory to END-dependent reprogramming will be investigated. In the first aim, a model positing that endoderm differentiation is directed by a sequentially redundant recursive series of feedforward interactions between the GATA transcription factors will tested by analyzing selected combinations of mutations in the pathway for synergistic effects on differentiation and behavior of endoderm cells. Feedback inhibition in the pathway and involvement of other endoderm-specific transcription factors will be examined. In the second aim, the hypothesis that activation and maintenance of a differentiated state is necessary to prevent cells from undergoing reprogramming and trans- differentiation will be tested. The role of intercellular communication in commitment of cells to specific differentiated states and the action of Notch signaling in the pluripotency AE commitment switch during embryogenesis will be evaluated. In the third aim, RNAi-based screens will be performed to identify components that regulate pluripotency and commitment both in the early embryo and in developing adult germline cells. The identified genes will be examined for cell type and temporal specificity in regulating competence for reprogramming. Collectively, these studies will reveal mechanistic steps that progressively restrict the developmental potential of differentiating cells. Results of these studies promise to advance our understanding of normal and defective animal development and may guide methods for creating new multipotential precursor cells from cells that are otherwise normally restricted in developmental potential, which can be used for generating new tissue and organs. PUBLIC HEALTH RELEVANCE: This project analyzes how specialized cells, such as neurons and gut cells, are prevented from becoming other types of cells, providing information about how stem cells can produce many different cell types. It will also shed light on how cells become the right type at the proper time and place during development, which is key to avoiding human birth defects.